Receptor of the small rhinovirus receptor group

ABSTRACT

A substantially pure receptor with binding activity for rhinoviruses of the small receptor group is disclosed, which has the following characteristics: 
     (a) a molecular weight of 120 kD on a polyacrylamide gel in the presence of SDS; 
     (b) a sedimentation constant, determined by sucrose gradient centrifugation in the presence of detergents, corresponding to about 28.4 S; 
     (c) is bound by Lens culinaris lectin; 
     (d) is not bound by heparin-sepharose; 
     (e) is bound irreversibly to an anion exchanger; 
     (f) has binding activity which is insensitive to neuraminidase; 
     (g) consists of sub-units connected by intermolecular disulfide bridges; 
     (h) shows no binding activity to rhinoviruses in the presence of EDTA; and 
     (i) has a binding activity to rhinoviruses which is only slightly influenced by iodoacetamide. 
     A receptor subunit of the above-described receptor, produced by complete reduction of said receptor, is also disclosed. Multimeric forms of the above-described receptor, obtained by controlled oxidation of the monomeric receptor, are also disclosed. Methods of providing protection against infection by rhinoviruses of the small receptor group with the substantially pure receptor of the present invention, are also disclosed. A method for the production of receptors for the small rhinovirus receptor group is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. application Ser. No. 08/095,246, filed Jul.22, 1993, now U.S. Pat. No. 5,304,636 which is a continuation of U.S.application Ser. No. 07/294,512, filed Feb. 14, 1989, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the receptor of the small receptor group ofhuman rhinoviruses, the purification and use thereof.

2. Brief Description of the Background Art

Human rhinoviruses constitute a large genus within the family of Picornaviruses and contain over 90 different serotypes Fox, J. P., American J.Epid. 103:345-354 (1976) and Melnick, J. L., Proc. Med. Virol.26:214-232 (1980). These RNA viruses affect the respiratory tract ofhumans and cause acute infections which may lead to colds, coughs,hoarseness, etc., and are generally known as colds Stott, E. J. et al.,Ann. Rev. Microbiol. 26:503-525 (1972). Infections caused byrhinoviruses are among the most common diseases in man. Although thecourse of the diseases is generally harmless, colds do neverthelessresult in general weakening of the organism. This may then give rise tosecondary infections caused by other pathogens.

The large group of human rhinoviruses can be subdivided into twosub-groups if the competition for binding sites on the cell surface inhuman cell culture cells (generally HeLa cells) is used as the criterionfor classification. This original classification of a fewrepresentatives of the rhinoviruses Lonberg-Holm, K. et al., Nature259:679-681 (1976) has been extended to 88 representatives as a resultof a wide range of experiments Colonno, R. J. et at., Journal ofVirology 57:7-12 (1986) and Abraham, G. and Colonno, R. J., Journal ofVirology 51:340-345 (1984). The result of these experiments was toindicate that in spite of the large number, there are surprisingly onlytwo different receptors on the cell surface to which representatives ofone or other group of rhinoviruses can bind. Up till now, 78 serotypesof the large "rhinovirus receptor group" and 8 of the small "rhinovirusreceptor group" have been classified (RVRG). 2 other representatives didnot behave clearly so that they could not be definitively classifiedAbraham, G. and Colonno, R. J., Journal of Virology 51:340-345 (1984).

In recent years, a considerable increase in rhinovirus infections hasbeen discovered in densely populated areas. Whereas the majority ofother infectious diseases result in a long-lasting or permanent immunityfrom the pathogen in question, infections caused by rhinoviruses mayrecur again and again. The reason for the absence of any lastingimmunity is the large variety of strains of rhinovirus which show littleor no immunological inter-reaction with one another Fox, J. P. Amer. J.Epidem. 103:345-354 (1976) and Melnick, J. L., Proc. Med. Virol.26:214-232 (1980). After infection has occurred, antibodies against thestrain of virus in question can be detected but these do not confer anyprotection against other rhinovirus strains. In view of the large numberof strains circulating in the population, repeated infections byrhinoviruses are possible.

Therefore, the presence of only two receptors offers promisingpossibilities for the successful combating of rhinovital infections.

Since receptors are generally highly specific, there is a possibility ofachieving controlled influence on the receptors by means of suitablesubstances, for example by blocking the receptors. If substances whichblock the receptor are used, the penetration of receptor-specificviruses into the cell can be prevented. The same substances which canprevent infection in this way can also be used for the treatment of amanifest rhinovirus infection. The production of such substances is madesubstantially easier and in some cases made possible for the first time,if the receptor in question is characterised.

SUMMARY OF THE INVENTION

One aim of this invention was therefore to isolate and purify thereceptor for the small RVRG.

The only information on rhinovital receptors available hitherto hasconcerned the receptor of the large RVRG.

The purification and characterisation of the receptor of the large RVRGwas effected using a monoclonal antibody obtained by immunizing micewith HeLa cells. This receptor is glycosylated and has a molecularweight of about 440 kD in its native state; denaturing with sodiumlaurylsulphate results in a dissociation into subunits of 90 kD, leadingone to conclude that the functional receptor is present as a pentamerTomassini, J. E. and Colonno, R. J., Journal of Virology 58:290-295(1986). Hitherto, the receptor for the small RVRG has neither beencharacterized nor purified. The only data on this receptor indicate itsprotein structure and also show that these or similar proteins are alsopresent on cells in a number of other species. This distinguishes thereceptor of the small RVRG essentially from the receptor of the largegroup, which has only been found in human cells and, in a few cases, inmonkey cells. Influencing of this receptor, for example blocking thereceptor with substances which prevent penetration of the virus into thecell, would appear to be suitable as a possible prevention or eventreatment for an existing infection.

DETAILED DESCRIPTION OF THE INVENTION

The aim of this invention was therefore to provide the prerequisites forpreparing substances which give protection against infections byrhinoviruses of the small receptor group.

This is achieved in the present invention by isolating the receptor fromcell membrane, for example HeLa cell membranes. These cells werecultivated in suspension by methods known per se, the cells were brokenup, the nuclei removed and the membranes purified. The receptors foundin the membranes were then solubilized.

To achieve optimum solubilization of active receptors from purified HeLacells, various detergents were tested at different concentrations. Thecritical factor in choosing a specific detergent was its ability tosolubilize as much membrane material as possible with the highestpossible virus binding activity. 1% 1-O-n-octyl-β-D-glucopyranosideproved to be the most suitable (FIG. 1A and 1B).

The insoluble constituents were removed and the receptors in solutionwere further purified. In order to be able to monitor the virus bindingactivity, a sensitive filter binding test was developed which makes itpossible for ³⁵ S-labelled virus to bind to receptors which had beenimmobilized on nitrocellulose paper.

The viruses required for the test were cultivated and purified in amanner known per se Skern, T. et al., Virology 136:125-132 (1984).

The receptors according to the invention were purified bychromatographic methods.

Since it is known that the majority of membrane proteins areglycosylated, the receptor was purified on a Lens culinaris lectincolumn. This lectin has specificity for α-D-glucose and α-D-mannoseunits Young, N. M. et al., J. Biol. Chem. 271:1596-1601 (1971). Boundmaterial was eluted with 1M α-D-methylglucoside in phosphate-bufferedNaCl solution with 1% octylglucoside.

Aliquots were applied in duplicate to nitrocellulose and incubated bothwith native and with heated virus. Autoradiography of the fractionsshowed strong binding to the native virus in the case of the materialwhich had been eluted, compared with the fractions from the materialwhich had run through. The heated virus showed weak binding to therun-through material. This indicates nonspecific interactions which arecaused by the high proportion of hydrophobic proteins:. heatedrhinovirus has greater hydrophobicity Lonberg-Holm, K. and Whiteley, N.M., Journal of Virology 19:857-870 (1976). Since it had been establishedthat, on being stored for a fairly long time at 4° C., purified virusgraduaIIy changes into particles which have the same antigenicity asheated virus (C-determinants), these contaminations were separated offby immunoprecipitation with C-determinant-specific monoclonalantibodies, for example mAK 2G2, immediately before the binding test wascarried out. These monoclonal antibodies are obtained in a manner knownper se by immunizing mice or rabbits with C-determinants andsubsequently cloning according to Kohler and Milstein, Nature256:495-497 (1975).

In addition to the L. culinaris lectin column, concanavalin A, ricin andheparin-Sepharose columns were also used to purify the receptor. Therun-through and eluted material were tested as above. Con.A Sepharosecolumns were eluted with 1 M α-D-methyl-mannoside, approximately 20% ofthe binding activity being recoverable; elution of the ricin column with1M galactose resulted in approximately 100% recoverable bindingactivity. By contrast with the receptor for the Coxsackie B virus groupKrah, D. L. and Crowell, R. L., Journal of Virology 53:867-870 (1985)heparin-Sepharose did not retard the binding activity.

The eluate from the L. culinaris column was separated on a Superosecolumn by FPLC (Pharmacia) (gel permeation chromatography). Bycomparison with market proteins, the molecular weight of the activereceptor was determined as 450 kD. At the same time, a substantialproportion of contaminating proteins could be removed (FIG. 2).

The minor group receptor migrates with an apparent molecular weight of120 kD on the polyacrylamide gel in presence of SDS. Sometimes a barelyvisible band with a slightly lower molecular weight can also be observedwhich might represent a modification of the bulk of the receptorprotein. The molecular weight of both forms of the receptor areconsiderably higher than found for the major group receptor (90 kD). Asboth proteins exhibit a molecular weight of about 450 kD in their nativestate it is not unlikely that their subunit structure is similar. Theactual molecular weight might however differ from the one determined bygel permeation chromotography because of the small difference ofretention volumes of proteins in this high molecular weight range. Thepicornavirus structure shows a deep depression (the canyon) runningaround the fivefold axes of icosahedral symmetry which is thought tocontain the receptor binding site Rossman, M. G. et al., Nature317:145-154 (1985). It has been proposed that the rhinovirus major groupreceptor and the receptor for the coxsackie B virus group Mapoles, J. E.et al., Journal of Virology 55:560-566 (1985) bind the virus at the fivefold axes. The question whether the minor group receptor is a Dentamerremains open as the molecular weight of its subunits is rather high whencompared to the major group receptor.

By sucrose gradient centrifugation, it was possible to determine thesedimentation constant of the receptor. For this purpose, L. culinarispurified receptor was applied to a sucrose gradient and centrifuged.

The activity peak was found to be at the position of the gradient whichcorresponds to the sedimentation constant of 28.4 S (FIG. 3).

Preliminary tests had shown that the receptor could no longer be elutedfrom an anion exchange chromatography column. Since the receptor isinsensitive to neuraminidase, sialic acid was removed from theglycoprotein in order to reduce the ionic interaction with the columnmaterial. The sample was then applied to a mono Q column (Pharmacia) andthe receptor was eluted with an NaCl gradient. The binding activitycould be detected as a broad peak at about 250 mM NaCl (FIG. 4).

It is also possible to purify the receptors according to the inventionby a combination of chromatographic purification steps on differentchromatography materials.

The chemical properties and structural requirements of the receptoraccording to the invention for vital binding were determined with theaid of enzymes and chemical reagents (Table 1).

Trypsin treatment destroys the binding activity entirely. This agreeswith known results from enzymatic treatment of cell surfaces Stott, E.J. and Heath, G. F., Journal of Gen. Virology 6:15-24 (1970) andindicates the protein nature of the receptor.

Treatment of the solubilized receptor with neuraminidase resulted, inreproducible manner, in a slight increase in the binding activity. Thistreatment may possibly lead to better accessibility of the region on thereceptor molecule which is the target of the virus interaction. As aresult, sialic acid is not necessary for the virus binding.Dithiothreitol (DTT) destroys the binding activity, leading one toconclude that disulphide bridges are involved in maintaining the correctfolding of the protein. The surprisingly high molecular weight,determined by gel permeation chromatography and gradient centrifugation,indicates an oligomeric structure for the receptor molecule. Thesensitivity to DTT might lead one to conclude that intermoleculardisulphide bridges are necessary for the association of the hypotheticalsub-units.

Treatment with iodacetamide reduces the binding activity only slightlyand indicates that free sulphydryl groups are not necessary forefficient binding.

In the presence of ethylenediaminotetraacetic acid (EDTA) duringincubation of the nitrocellulose filters with ³⁵ S-labelled virus, nobinding could be detected. This agrees with earlier investigations whichshowed the need for the presence of divalent cations for interaction ofthe rhinoviruses with the cell surface Noble-Harvey, J. andLonberg-Holm, K., Journal of Gen. Virology 25:83-91 (1974).

Competitive binding assays between pairs of serotypes had been used inorder to classify the human rhinoviruses into the two receptor classesLonberg-Holm, K. et al., Nature 259:679-681 (1976) and Abraham, G. andColonno, R. J., Journal of Virology 51:340-345 (1984). In the presentinvention, therefore, HRV2 and HRV49 were used as representatives of thesmall receptor group and HRV89 as representative of the large receptorgroup in competitive experiments to discover the specificity of thereceptors according to the invention. The nitrocellulose filters withimmobilized receptors were incubated in the presence of an approximately20-fold excess of either HRV2 or HRV89 with labelled HRV2. As shown inTable 2, the binding was massively suppressed in the presence ofnon-labelled HRV2 but unaffected by HRV89. In order to check theseresults, the tests were repeated with labelled HRV49, using HRV2 andHRV89 as competitors. Once again, it is obvious that HRV2 reducesbinding on a massive scale but HRV89 has no effect.

Although HRV2 and HRV89 bind to different receptors, their capsidproteins are surprisingly similar Duechler, M. et al., Proc. Natl. Acad.Sci. (in Press) (1987). A detailed comparison of structure between HRV2and HRV14, based on the X-ray structure analysis of HRV14, was recentlyset up Blaas, D. et al., Proteins (in Press) (1987). Both HRV14 andHRV89 bind to the receptor of the large RVRG. It can therefore beexpected that a cluster of preserved amino acids will be found at thehypothetical receptor binding site. Up till now, however, it has not yetbeen possible to discover a simple pattern of conservative amino acidswithin the Canyon region.

The present invention makes it possible for the first time to producereceptors for the small RVRG.

Using the receptors according to the invention it is possible for thefirst time to carry out controlled investigations on the virus/receptorinteractions. Of particular importance is the locating of the regions onthe receptors which are finally responsible for the viral activity. Oncethese areas are known, it should be possible to produce substances whichare directed specifically against these areas, and thereby possiblyblock the receptors for a variety of different rhinoviruses.

The present invention relates to receptors which can be prepared by theprocess described and which bind representatives of the small RVRG.

The present invention also relates to the receptors which can beproduced from the natural receptors by methods known to those skilled inthe art. By way of example, there may be mentioned the sub-units of thenatural receptor, which are obtained by treating with reducing agents,and which can be purified for example by electrophoretic methods. Thesesub-units may be used, for example, to produce polyclonal and/ormonoclonal antibodies which can be used preparatively, diagnosticallyand/or therapeutically in a similar manner to the correspondingantibodies against the natural receptors. The receptor sub-units mayalso be used in a similar manner to the natural receptors.

The present invention also relates to the modifications of the naturalreceptors and/or the sub-units thereof which can be obtained bycontrolled enzymatic treatment. As has been shown in this invention,treatment with trypsin destroys the binding activity of the receptorsaccording to the invention, while neuraminidase caused a slight increasein activity. Therefore it is conceivable, and anyone skilled in the artcan check this in a non-inventive manner, that specific enzymes and/orchemical reagents result in receptors which either have an improvedactivity and/or are easier to apply and use and/or have better stabilitycompared with the natural receptors. These modifications may, forexample, result in parts of the protein chain being severed or cut outand/or the protein chains being cut up, in all or some of the sub-unitsof the natural receptors.

In addition to these modificetions it is also possible to convert thenatural receptors either wholly or partially into the sub-units, forexample by controlled reduction. These large or small sub-units may alsobe linked together, for example by controlled oxidation, to form largeor small units which are rearranged compared with the natural receptors.

Suitable reducing agents for cleaving disulphide bridges include, forexample, thiol compounds such as thiophenol, 4-nitrothiophenol,1,4-butanedithiol and particularly 1,4-dithiothreitol. The reduction isadvantageously carried out in an aqueous/alkaline medium, for example inthe dilute aqueous solution of an alkali metal hydroxide, e.g. sodiumhydroxide, alkali metal carbonate, e.g. sodium carbonate or an organicbase, more particularly a tri-lower alkylamine, e.g. triethylamine, atambient temperature.

Suitable oxidizing agents for the re-linking of disulphide bonds in thereduced polypeptides include, for example, oxygen from the air, which ispassed through an aqueous solution of the polypeptide to which acatalytic amount of a transition metal salt, e.g. iron(III)-sulphate,iron(III)-chloride or copper(II)-sulphate, may have been added; iodine,including iodine in the form of the potassium iodide adduct KI₃, whichis preferably used in alcoholic, e.g. methanolic, or aqueous-alcoholic,e.g. aqueous-methanolic solution; potassium hexacyanoferrate(III) inaqueous solution; 1,2-diiodoethane or dimethyl or diethylazodicarboxylate, which are reacted in water or in a mixture consistingof water and a water-miscible alcohol, e.g. methanol. Oxidation is moreparticularly carried out at ambient temperature.

The removal of the reagents, particularly the salts and the oxidants andreducing agents and their secondary products, from the desired compoundis carried out by methods known per se, for example by molecular weightfiltration, e.g. on Sephadex or Biogel.

All the modifications may be used in the same way as the naturalreceptors according to the invention. The products obtainable in thisway, such as the antibodies, like the modifications, fall within thescope of the present invention.

The receptor according to the invention is soluble, so that it is easyto handle and use.

However, it is also possible to bind the receptors to a solid carrierand to use them in this form for diagnostic and preparative purposes.Suitable carriers include all the usual solid carriers such aspolystyrene, glass, dextrans and also biological membranes and lipidvesicles.

It is also possible to bind conventional labels to the receptors and touse them in this form for diagnostic purposes. It is also possible touse the receptors for the therapeutic treatment of viral infections. Ifthe receptors according to the invention are bound to a carrier, theymay be used both diagnostically and also preparatively to bind the vitalprotein, for example by means of so-called affinity chromatography.Diagnostically, a vital protein can be detected in the usual way by areceptor bound to a carrier, e.g. by means of antibodies or labelledantibodies. The labelling used may be, for example, radioactivelabelling, an enzyme or a fluorescent group.

When the receptors according to the invention are used therapeutically,they may be ejected in suitably highly refined-form, so that they canthen inhibit competitively against the natural receptor. Preferably,soluble receptors will be used for this purpose. Such solutions may alsobe used for diagnosis and differential diagnosis.

An exceptionally important application is the use of the receptorsaccording to the invention for producing polyclonal and/or monoclonalantibodies which act specifically against the receptors located in thecell membranes. Antibodies of this kind may first of all be useddiagnostically to show up and determine the receptors on cells orbiological cell material. Furthermore, they may be used therapeuticallyto block the receptors in the cell membranes. Consequently, they open uptotally new methods and possibilities.

Further, the invention relates to a hybrid cell line which secretesmonoclonal antibodies against a receptor with binding activity forrhinoviruses of the small receptor group. The receptor against which thehybrid cell lines secrete monoclonal antibodies., has a molecularweight, determined by gel permeation chromatography, of about 450kilodaltons, and a sedimentation constant, determined by sucrosegradient centrifugation in the presence of detergents, corresponding toabout 28.4 S. The receptor is bound by Lens culinaris lectin, but is notbound by heparin-sepharose; yet, it binds irreversibly to an anionexchanger. Further, it has a binding activity which is insensitive toneuraminidase. It consists of sub-units associated by intermoleculardisulfide bridges, shows no binding to rhinoviruses in the presence ofEDTA, and has a binding activity to rhinoviruses which is only slightlyinfluenced by iodacetamide. Moreover, the invention relates to hybridcell lines which secret monocional antibodies against a receptorsub-unit produced by complete reduction of a receptor with propertieswhich are described above. Further, the receptor may be comprised of atleast two of the receptor sub-units, as mentioned above, and which isnot the natural receptor. The receptor may be produced by controlledreduction of a receptor, as described above. Furthermore, it may beprepared from a receptor, as described above, by controlled treatmentwith one or more enzymes and/or chemicals mentioned above. Finally, thereceptor may be produced by controlled oxidation of at least two of thereceptor sub-units mentioned above.

A further aspect of tile invention relates to a process for preparingmonoclonal antibodies which specifically neutralize wholly or partly areceptor, as described above, or which specifically bind to one of tileabove-mentioned receptors. In said process, host animals are immunizedwith a receptor, as described above, B-lymphocytes from said hostanimals are fused with myeloma cells, the hybrid cell lines secretingsaid monoclonal antibodies are subcloned and cultivated in vitro or invivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A Filter binding test according to example 4. Receptor protein wasapplied to a nitrocellulose membrane. After saturation of non-specificprotein binding sites with 2% bovine serum albumin (BSA) in PBS-bufferthe filter was incubated with ³⁵ S-methionine labelled native HRV2 inthe presence of various detergents (SDS, Triton X-100, CHAPS,Zwittergent, Octylglycoside or DOC).

FIG. 1B Filter binding test showing a negative control. Correspondingfilter binding test with ³⁵ S-methionine labelled denatured HRV2.

FIG. 2 Gel permeation chromatography of the solubilized receptor on aSuperose 6 HR 10/30 column. Membranes equivalent to 10⁸ cells weresolubilized in OG (15 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 mMMgC₂, 1 mM CaCl₂ (PBS) with 1% octyl glucoside), applied to a 1 ml L.culinaris column and the adsorbed material was eluted with 1Mα-D-methylglucoside in OG. The eluate was concentrated in a Centricontest tube to 0.5 ml and applied to the Superose column. The column wasdeveloped with 0.2 ml/min OG and 0.5 ml fractions were collected. Thebinding activity of the individual fractions, the positions of themarker proteins (determined in a separate experiment) and the extinctionat 280 nm are shown.

FIG. 3 Sucrose gradient centrifugation of the solubilized receptor.Material which had been eluted from an L. culinaris column wasconcentrated (see the legend to FIG. 2) and separated on a 10-40%sucrose gradient in OG. The binding activity of the fractions (0.4 ml),the position of the marker proteins catalase (Cat) and aldolase (Ald)and the extinction at 280 nm are shown.

FIG. 4 Mono Q anion exchange chromatography. ractions 14 to 16 of thegel permeation chromatography (FIG. 2) were treated with neuraminidaseand the material was separated by FPLC on mono Q. The binding activityof the individual fractions (0.5 ml), the extinction at 280 nm and thepath of the gradient are shown.

Materials

1-O-n-Octyl-β-glucopyranoside, Tween 40 and3-(3-cholamidopropyl)-dimethylammonio-1-propane sulphonate (CHAPS) wereobtained from Sigma, N-tetradecyl-N,N-dimethyl-ammonio-3-propanesulphonate (Zwittergent 3-14) was obtained from Serra. The otherdetergents came from Merck, trypsin from Miles and ³⁵ S-methionine (1350Ci/mmol) from Amersham.

EXAMPLE 1 Preparation of the viruses

HRV2, HRV49 and HRV89 were cultivated essentially as described in HeLacell suspension and then purified Skern, T. et al., Virology 136:125-132(1984). The cultivation, isolation and purification of HRV2 will bedescribed here by way of example.

HeLa cells (strain HeLa Ohio, 03-147, Flow Laboratories, England) weregrown in suspension at 37° C. The suspension medium (Thomas, D.C.,Conant, R. M. and Hamparian, V.U., 1970, Proc. Soc. Exp. Biol. Med. 133,62-65; Stott, E. J. and Heath, G. F., 1970, J. Gen. Virol, 6, 15-24)consisted of a Joklik modification of MEM for suspension (Gibco072-1300) and 7% horse serum (Seromed 0135). The inoculation density was5-10×10⁴ cells/ml and the volume was 500 ml. The suspension wascentrifuged at a cell density of 1×10⁶ cells/ml under sterile conditionsat 300 g for 10 minutes. The supernatant was removed by suctionfiltering and the cells were resuspended in 100 ml of infection medium(Joklik modification of MEM for suspension culture with 2% horse serumand 2 mM MgCl₂). By carefully sucking up several times in a 20 mlpipette, the cells were homogeneously distributed in the infectionmedium. The medium was then made up to 500 ml. The cell suspension wasbrought to 34° C. and infected with HRV2 (twice plaque-purified) at amultiplicity of 0.1 viruses per cell. The HRV2 strain was obtained fromthe American Type Culture Collection, (ATCC VR-482 and VR-1112). Thestrain used was neutralized with antiserum against HRV2 (American TypeCulture Collection, Cat. No. ATCC VR-1112 AS/GP). The control serum usedwas an antiserum against HRV7 (Cat. No. ATCC VR-1117 AS/GP) which showedno neutralization. After 60 hours at 34° C. the virus was harvested.Virus was obtained both from the cells and cell fragments and also fromthe medium.

For this purpose, the medium was separated from infected cells and cellfragments by centrifuging for 10 minutes at 1500 g and then suctionfiltering. The precipitate was frozen at -70° C.

The cell precipitates from 12 liters of suspension culture werecombined, resuspended in 40 ml of TM buffer (20 mM Tris/HCl, pH 7.5, 2mM MgCl₂), put on ice for 15 minutes, then broken up in a Douncehomogenizer and the mixture was centrifuged for 30 minutes at 6000 g.The precipitate was then washed once again in 10 ml of TM buffer. Thetwo supernatents were combined and centrifuged for 3 hours at 110,000 gin order to pellet the virus. The virus pellet was then taken up in 10ml of KTMP buffer (50 mM KCl, 50 mM Tris/HCl, pH 7.5, 5 mM MgCl₂, 2 mMmercaptoethanol, 1 mM puromycin, 0.5 mM GTP) and after the addition of150 mcg of DNase I (Sigma, ribonuclease-free) it was incubated for 1hour on ice.

The virus was precipitated from the infection medium with stirring at 4°C. with polyethylene glycol 6000 (PEG 6000; Merck) at a concentration of7% and 450 mM NaCl (Korant, B. D., Lonberg-Holm, K., Noble, J. andStasny, J. T., 1972, Virology 48, 71-86). After 4 hours in the cold, thevirus was centrifuged off for 30 minutes at 1500 g, the precipitate wasresuspended in 10 ml of KTMP buffer containing 75 mcg of DNase I, themixture was incubated for 1 hour on ice and then frozen at -70° C.

The virus suspensions obtained from the cells and from the medium werecombined, incubated for 5 min. at 37° C., cooled by the addition of 60ml of cold TE buffer (10 mM Tris/HCl, pH 7.4, 1 mM EDTA) and thensonicated for 5 min in an ice bath.

The suspension was then centrifuged for 30 minutes at 6000 g. 920 ml ofTE buffer containing 7% PEG 6000 and 450 mM NaCl were added to thesupernatant, this was stirred carefully for 4 hours at 4° C. and theprecipitate formed was pelleted for 30 minutes at 6000 g. Theprecipitate was once again taken up in 100 ml of TM buffer, the viruswas precipitated as above by the addition of PEG 6000 and NaCl andpelleted. The precipitate was resuspended in 40 ml of TM buffer, thesuspension was centrifuged for 30 minutes at 6000 g and the virus waspelleted for 3 hours at 110,000 g. The precipitate was dissolved in 1 mlTM buffer, incubated for 1 hour at 4° C. after the addition of 50 mcg ofDNase I and then 1 ml of TE buffer was added. For further purification,the virus suspension was centrifuged on sucrose gradients (10-30% w/w inTE buffer) for 4 hours at 4° C. and at 110,000 g. From the extinction at260 nm, the fractions containing the virus were discovered and dilutedwith TM buffer so that the final sucrose concentration was 10%. Thencentrifuging was carried out for 8 hours at 85,000 g.

The virus pellet was taken up in 1 ml TM buffer and stored at -70° C. Tocheck the purity of the virus preparation, electrophoresis was carriedout on a 2.5% polyacrylamide gel in the presence of 0.1% sodiumdodecylsulphate (Laemmli, U. K., 1970 Nature (London) 277, 680-685) andthe protein bands were stained with Coomassie Brilliant Blue.

EXAMPLE 2 Preparation of ³⁵ S-methionine-labelled human rhinovirusserotype 2 (HRV2)

2 HeLa cell mono layers in 165 cm² Petri dishes were infected with anMOI (Multiplicity of Infection) of 40 with HRV2 for 1 hour at 34° C. inmethionine-free MEM medium (Gibco) with 2% dialyzed fetal calf serum(Flow). The cells were washed twice with PBS and incubation wascontinued at 34° C. in fresh medium. After 3 hours, 1 mCi ³⁵S-methionine (1350 Ci/mmol, Amersham) was added to each mono layer andincubation was continued for a total of 24 hours. The medium of infectedcells and cell fragments was separated by 10 minutes centrifuging at1500 g and suction filtered. The precipitate was frozen at -70° C. in 5ml of 10 mM Tris, 10 mM EDTA, pH 7.5 (Tris/EDTA) and thawed again. Thesupernatant and frozen/thawed precipitate were combined and centrifugedfor 30 min. at 45,000×g. The supernatant from this centrifugation wascentrifuged for 2 hours at 140,000×g. The virus pellet was resuspendedin 300 mcl Tris/EDTA and the virus was purified over a 10-30% sucrosegradient as above. The individual fractions of the gradient wereanalysed by SDS electrophoresis in 12% polyacrylamide gels. The purevirus fractions were combined and stored in the presence of 1% BSA(bovine serum albumin) at 4° C. for at most 4 weeks.

In order to remove virus with an altered capsid structure from thepreparations from Examples 1 and 2, 20 mcl of immunoadsorbant,containing monoclonal antibodies against the C-determinant, for examplemAK 2G2, were incubated with the purified virus for 30 min. and pelletedbefore the vital probes were removed.

Preparation of the immunoadsorbant

Staphylococcus aureus (BRL) cells were suspended as a 10% w/w suspensionin water. The cells were washed twice with PBS, 1/5 volrabbit-anti-mouse IgG serum (Behring) was added; the suspension wasincubated for 1 hour at ambient temperature. The cells were washed twicewith PBS and incubated again for 1 hour with 1/5 vol mouse as-citesfluid, which contained monoclonal antibodies against the C-determinants(e.g. mAK 2G2). After washing twice, it was pelleted, the pellet wasresuspended in PBS (10% w/w) and the preparations of the radioactiveviruses were added.

EXAMPLE 3 Solubilization of the receptor from HeLa cells

HeLa cells (strain HeLa-Ohio, 03-147, Flow Laboratories, England) werecultivated in suspension at 37° C. The suspension medium (Thomas, D. C.,Conant, R. M. and Hamparian, V. U., 1970, Proc. Soc. Exp. Biol. Med.133, 62-65; Stott, E. J. and Heath, G. F., 1970, J. Gen. Virol. 6,15-24) consisted of a Joklik modification of MEM for suspension (Gibco072-1300) and 7% horse serum (Seromed 0135). The inoculation density was5-10×10⁴ cells/ml and the volume was 500 ml. The suspension wascentrifuged at a cell density of 1×10⁶ cells/ml under sterile conditionsat 300 g for 10 min. The supernatant was removed by suction filteringand the cells were washed twice with phosphate-buffered saline solution(PBS). 10⁹ cells were suspended in 20 ml of isotonic buffer (10 mMHEPES-KOH, pH 7.9, 140 mM KCl, 1.5 mM MgCl₂, 0.5 mM EDTA, 0.2 mMphenylmethylsulphonylfluoride) and broken up in the cold with 200 pulsesof a 50 ml Dounce homogenizer. Cell nuclei were removed by centrifugingfor 3 minutes at 1000×g. The membranes were then further purified by thetwo-phase method Brunette, D. M. and Till, J. E., J. Membr. Biol.5:215-224 (1971). Membranes corresponding to 2×10⁸ cells per ml weretaken up in PBS and stored in liquid nitrogen. To solubilize them, 2×10⁸cell equivalents were suspended in 1 ml of 1% octylglucoside in PBS (OG)and any insoluble material was removed by centrifuging at 80,000×g for 1hour. The supernatant was used for column chromatography.

EXAMPLE 4 Filter binding test

Fractions from column chromatography to be tested for activity wereapplied to a nitrocellulose membrane (BA85, Schleicher and Schull) in adot-blot apparatus (Bio-Rad). The probes were left to seep in at ambienttemperature. Then liquid residues were suction filtered under a gentlewater jet vacuum and non-specific protein binding sites were saturatedwith 2% bovine serum albumin (BSA) in PBS at 4° C. overnight. Thefilters were then incubated with 10⁵ cpm ³⁵ S-methionine labelled HRV2in 1% Tween 40, 0.5% sodium deoxycholate and 10 mM(3-(3-cholamidopropyl)-dimethylammonio-1-propane sulphonate) in PBS for1 hour. The membranes were washed twice with 2% BSA in PBS, then driedand the round areas corresponding to the probes were stamped out; theradioactivity was measured in a liquid scintillation counter.

As a specificity control, HRV2 was heated to 56° C. for 10 minutesbefore the incubation of the nitrocellulose filters LonbergHolm, K. andYin, F. H., Journal of Virology 12:114-123 (1973). After this treatment,no binding to any of the probes could be detected (FIG. 1B). From thisit was concluded that the binding of native HRV2 to the immobilizedmaterial can actually be ascribed to a specific interaction of the viruswith the receptor.

EXAMPLE 5 Affinity chromatography on Lens culinaris lectin columns

10⁸ cell equivalents were solubilized as described and applied to an L.culinaris column (1 ml) equilibrated with OG. The column was washed with5 ml of OG and bound material was eluted with 2 ml of 1 Mα-D-methyl-gluceside in OG. The binding test showed that almost 100% ofthe binding activity could be recovered, whereas about 90% of the totalprotein had been removed.

EXAMPLE 6 Gel permeation chromatography

The eluate from the L. culinaris column was concentrated down to 0.5 mlwith a Centricon tube (exclusion 30 kD) and seoarated on a Superose 6 HR10/30 column (equilibrated with OG-buffer) by FPLC (Pharmacia). Bycomparison with marker proteins the molecular weight of the activereceptor could be determined as 450 kD. At the same time a largeproportion of contaminating proteins could be removed (FIG. 2).

EXAMPLE 7 Sucrose gradient centrifugation

L. culinaris purified receptor (as above) was applied to a sucrosegradient (10-40% in OG) and centrifuged for 8 hours at 38 krpm at 4° C.The activity peak was found at the position on the gradientcorresponding to the sedimentation constant of 28.4 S. The positions ofthe marker proteins were determined in another gradient (FIG. 3). As aresult of the presence of detergents, the markers sedimented atcalculated sedimentation coefficients of 15.0 S and 21.9 S (7.3 S and11.3 S in the absence of detergents).

EXAMPLE 8 Anion exchange chromatography

It had been found in preliminary tests that the receptor could no longerbe eluted by mono Q HR 5/5 columns (Pharmacia). Therefore, receptorwhich had been subjected to preliminary purification using L. culinarisand gel permeation was treated with 1 U neuraminidase/mg of protein for60 min at 37° C. in order to remove the strongly acidic neuramino acidgroups. The sample was then diluted with 10 mM sodium phosphate buffer,pH 7, 1% octylglucoside, to twice the quantity and applied to a mono Qcolumn. The column was developed with a gradient of 0 to 1M NaCl in thesame buffer. The binding activity could be detected as a broad peak atabout 250 mM NaCl (FIG. 4).

EXAMPLE 9 Isolation and purification of the receptor

Plasma membranes of 2×10⁹ HeLa cells were solubilized in 5 ml PBScontaining 1% w/v 1-0-n-octyl-β-D-glucopyranoside and 0.01% w/v each ofL-α-p-tosyl-L-lysinechloromethyl ketone (TLCK) ,L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), andphenylmethyl-sulphonylfluoride (PMSF) (all from Sigma) for 10 minutes atroom temperature. Insoluble material was removed by centrifugation at 30krpm for 30 minutes in the Beckman 65 fixed angle rotor. The supernatantwas applied onto a 25 ml L. culinaris lectin column equilibrated with OG(PBS, 1% w/v octyl-glucoside) and bound material was eluted with 5 ml ofOG containing 1M α-methyl glucose. The eluate was brought to 50%saturation by addition of the same volume of saturated ammonium sulfate.The precipitated material was dissolved in 2 ml of buffer A (10 mMTris-HCl (DH 7.5), 5 mM EDTA, 1% w/v octyl-glucoside) and injected ontoa Mono P anion exchange column connected to a Pharmacia FPLC system. Theproteins were separated using a gradient from 0 to 100% buffer B (as Abut containing 1.5M MACl). Fractions containing the virus bindingactivity were pooled, concentrated with a centricon tube to 0.5 ml andrun on a Superose 6 column equilibrated with OG containing 5 mM EDNA.The protein concentration was monitored by the absorbance at 280 nm

The fractions from this column were concentrated to 50 μl, made 0.1% w/vSOS and run in triplicate on a 6% polyacrylamide gel containing 5 mMEDTA Laemmli, U. K., Nature (London) 277:680-685 (1970). The proteinsseparated in the gel were then either stained with Coomassie blue orelectrophoretically transferred to a nitrocellulose sheet Burnette, W.N., Analytical Biochemistry 112:195-203 (1981) which was incubated with4×10⁵ cpm ³⁵ S-labelled HRV2 under conditions as described for the dotblots. The nitrocellulose was then dried and autoradiographed. As acontrol for specific binding an identical blot was incubated in presenceof a 20 fold excess of unlabelled HRV2. It can be seen that fractions 6and 7 from the Superose column contained material which was able to bindHRV2 when transferred to the nitrocellulose. The autoradiograph showsseveral bands with an apparent molecular weight greater than 300 kD inaddition to one band at a position corresponding to approximately 120 kDwhen compared to protein markers run on the same gel. Only this 120 kDband disappears in the control containing excess unlabelled virusdemonstrating specific interaction of this protein with HRV2. Thepolyacrylamide gel containing identical samples and stained withCoomassie blue shows a very faint band at a position corresponding tothe radioactive band on the Western blot. This band is only found in thesamples obtained from fractions 6 and 7 which exhibit virus bindingactivity.

EXAMPLE 10 Binding tests

The restoration of an active receptor is dependent on mild conditions asboiling in SDS irreversibly destroys its activity. When the receptorpreparation was incubated with 10 mM dithiothreitol prior to loadingonto the polyacrylamide gel, no binding was observed. As the specificinteraction of rhinoviruses with their receptors is dependent on thepresence of divalent cations Noble-Harvey, J. and Ionberg-Holm, K.,Journal of Gen. Virology 25:83-91 (1974) a blot obtained from a sampleidentical to the one applied onto lane 1 was incubated with virus inpresence of EDNA. Under these conditions no binding could be observed.As further control an incubation of the nitrocellulose sheet with HRV2which had been heated at 56° C. was carried out. This treatment leads toa structural change of the vital capsid which precludes recognition ofthe virus on the HeLa cell surface Lonberg-Holm, K. and Yin, F. H.Journal of Virology 9:29-40 (1973). No binding occurred under theseconditions.

                  TABLE I                                                         ______________________________________                                        Sensitivity of the small rhinovirus group receptor                            Pretreatment of the solubilized                                                                   Binding assay                                             small receptor group                                                                              (% Bind.-act.)                                            ______________________________________                                        No pretreatment     100                                                       10 mcg trypsin       6                                                        50 mU neuraminidase 170                                                       10 mM dithiothreitol                                                                              15                                                        10 mM iodacetamide  80                                                        10 mM sodium periodate                                                                            70                                                        10 mM EDTA.sup.a     5                                                        ______________________________________                                         All preincubations were carried out at 37° C. for 30 minutes.          Note: .sup.a No pretreatment; instead, incubation was carried out with        labelled HRV2 in the presence of 10 mM EDTA.                             

                  TABLE 2                                                         ______________________________________                                        Competition between various rhinovirus serotypes                              for the small receptor group.                                                                  Binding assay                                                                 (% Bind.-act.)                                               ______________________________________                                        Competition of radioactively                                                  labelled HRV2 with:                                                           nothing            100                                                        HRV2               13                                                         HRV89              95                                                         Competition of radioactively                                                  labelled HRV49 with:                                                          nothing            100                                                        HRV2               15                                                         HRV89              90                                                         ______________________________________                                         The filters were incubated with a 20fold excess of nonlabelled virus as       described.                                                               

We claim:
 1. A method of detecting a rhinovirus of the small receptorgroup, said method comprising:(a) binding a receptor to a solid support,thereby obtaining a bound receptor, wherein said receptor has thefollowing characteristics:(i) a molecular weight of 120 kD on apolyacrylamide gel in the presence of SDS; (ii) a sedimentationconstant, determined by sucrose gradient centrifugation in the presenceof detergents, corresponding to about 28.4 S; (iii) is bound by Lensculinaris lectin; (iv) is not bound by heparin-sepharose; (v) bindsirreversibly to an anion exchanger; (vi) has binding activity which isinsensitive to neuraminidase; (vii) consists of sub-units connected byintermolecular disulfide bridges; (viii) shows no binding activity torhinoviruses in the presence of EDTA; and (ix) has a binding activity torhinoviruses which is only slightly influenced by iodacetamide: (b)contacting the bound receptor obtained in step (a) with a samplecomprised of said rhinovirus, thereby obtaining rhinovirus bound to saidreceptor; and (c) detecting said rhinovirus bound to said receptor,obtained in step (b), with a detectably labelled antibody directedagainst said rhinovirus.
 2. An antibody which specifically binds to areceptor, wherein the receptor has the following characteristics:(i) amolecular weight of 120 kD on a polyacrylamide gel in the presence ofSDS; (ii) a sedimentation constant, determined by sucrose gradientcentrifugation in the presence of detergents, corresponding to about28.4 S; (iii) is bound by Lens culinaris lectin; (iv) is not bound byheparin-sepharose; (v) binds irreversibly to an anion exchanger; (vi)has binding activity which is insensitive to neuraminidase: (vii)consists of sub-units connected by intermolecular disulfide bridges;(viii) shows no binding activity to rhinoviruses in the presence ofEDTA; and (ix) has a binding activity to rhinoviruses which is onlyslightly influenced by iodacetamide.
 3. The antibody of claim 2, whereinsaid antibody is a monoclonal antibody.
 4. A hybrid cell line whichproduces the monoclonal antibody of claim
 3. 5. A method of identifyinga receptor which specifically binds to rhinoviruses of the smallreceptor group in a sample, said method comprising adding to said samplethe monoclonal antibody of claim 3, wherein said monoclonal antibody isdetectably labeled.